Dengue virus and other flaviviruses including West Nile virus, tick-borne encephalitis virus, and Yellow Fever virus, are widespread, mosquito- or tick-borne human pathogens. About 40% of the worlds population lives in areas with substantial risk of dengue transmission, and as many as 100 million people may be infected annually, experiencing dengue fever and potentially lethal severe dengue. Development of a vaccine effective against all four dengue serotypes has been difficult because infection with one ofthe four serotypes does not lead to protective immunity against any of the other three and may lead to enhanced risk of severe, life-threatening illness, especially in children. Antivirals against dengue are of interest due to their potential to reduce the severity of dengue-associated disease as well as to reduce transmission. This proposal will contribute to efforts to develop dengue antivirals by developing mechanistically well-characterized small molecule inhibitors of dengue entry and using these to validate the dengue E protein as an antiviral target in vitro and in vivo. We have previously identified three structurally distinct lead compound series and inhibitors that exhibit a spectrum of activities against dengue virus entry. In the proposed work, we will use a combination of x-ray crystallography, medicinal chemistry, direct protein-small molecule affinity measurements, single-virion fusion and live-cell imaging assays, and resistance studies to optimize the pharmacological activity of our compounds and to elucidate the biochemical and structural mechanisms of action that correspond to potent, pan-serotype inhibition of dengue. Key to these efforts are quantitative assays, analogous to enzymatic activity measurements, for studying the biochemical functions of the viral envelope protein and that allow us to link the biochemical mechanism of small-molecule inhibitors with the step(s) in the cell-entry pathway at which they block infection. Optimized dengue entry inhibitors will be tested in a murine model of dengue infection and pathogenesis. Collectively, our efforts are aimed at producing compounds that can be advanced as preclinical candidates for dengue drug discovery efforts. More broadly, this work establishes the template for a rational approach to the development of small molecule inhibitors of other enveloped viruses as potential antivirals